Structural and functional analysis of ribosome initiation and ribosomal frameshifting.

核糖体起始和核糖体移码的结构和功能分析。

• 批准号: BB/G008051/1
• 负责人: Robert Gilbert
• 金额: $ 61.38万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG008051%2F1
• 关键词: Structural; functional; analysis; ribosome; initiation

Proteins are polymers of amino acids. The order of the amino acids is governed by the genetic code in the genes of the cell's DNA. In protein synthesis, a gene is first copied into messenger RNA (mRNA) - a single-strand copy of the DNA which retains the code - and then taken to the ribosome. Ribosomes are protein synthesising machines some 25-35nm in diameter and made up of two subunits, one large, one small. The mRNA is loaded linearly between the subunits, whereupon adaptor molecules called transfer RNAs (tRNAs) arrive to decode the mRNA. Each transfer RNA has an amino acid at one end, and at the other, a region which recognises a specific run of three consecutive nucleotides in the mRNA code, a 'triplet'. During protein synthesis, the ribosome moves along the mRNA triplet by triplet and at each step, a tRNA with the appropriate recognition sequence is recruited and the ribosome transfers the tRNA's amino acid to the growing chain of amino acids, the polypeptide. This project will study three aspects of protein synthesis. The first is how mRNA is recruited to the ribosome and fed between the subunits. In mammals, the mRNA interacts with the isolated small subunit of the ribosome in complex with a host of other proteins termed initiation factors. This initiation complex moves along the mRNA, scanning the sequence until the specific triplet that signifies the start of protein synthesis is identified. At this point, the large subunit joins and amino acid polymerisation begins. We wish to characterise in detail the structure of this scanning complex (termed the 48S complex), by purifying initiation complexes paused in the act of scanning along the mRNA, or at the initiation triplet, and study them using microscopy. Our microscope uses electrons rather than light, and is extremely powerful because the short wavelength of electrons allows the visualisation of fine molecular detail. The samples are also frozen at -180C, which keeps them in a natural and stable state, and so the technique is known as 'cryo-electron microscopy' (cryo-EM). The images we obtain will show us the structure of the 48S complex and tell us something of how the various initiation factors allow the small subunit to scan along the mRNA and to start protein synthesis. The second aspect is elongation, when amino acids are added, via tRNAs, to the growing chain. Inside the ribosome are multiple binding sites for tRNAs. A tRNA comes in at one site, binds to its complementary mRNA triplet and transfers its amino acid to the growing chain, moves to an adjacent site to make space for the next tRNA, and finally leaves the ribosome via an exit site. The movement of tRNAs has been hard to analyse at a molecular level. We have identified an mRNA signal termed a pseudoknot which blocks the progress of the ribosome along the mRNA and stalls the ribosome at the point when the tRNAs are moving within the ribosome. We will use cryo-EM to study the structure of ribosomes stalled at a pseudoknot to elucidate the molecular details of tRNA movement. Pseudoknots can also cause the ribosome to change reading frame, that is, to shift from reading one set of triplets (one frame) to reading an overlapping set (another frame), and this leads to a completely new amino acid sequence in the protein. Cryo-EM studies of pseudoknot-stalled ribosomes may thus be informative as to how the ribosome maintains the correct reading frame. The final aspect of the project concerns the structure of ribosomes prepared from mammalian cells. Because the ribosome plays a major role in controlling the rate of protein synthesis, and even which proteins are made, in different cell states the ribosome becomes modified in a variety of ways that affect which proteins are made and how rapidly. By determining structures for ribosomes from cells grown under known conditions we will make the first moves in showing the relationship between ribosome mechanism and regulatory processes in living cells.

蛋白质是氨基酸的聚合物。氨基酸的顺序由细胞 DNA 基因中的遗传密码控制。在蛋白质合成中，基因首先被复制成信使 RNA (mRNA)——保留密码的 DNA 的单链拷贝——然后被带到核糖体。核糖体是一种蛋白质合成机器，直径约为 25-35 nm，由一大一小两个亚基组成。 mRNA 在亚基之间线性加载，随后称为转移 RNA (tRNA) 的接头分子到达解码 mRNA。每个转移RNA一端有一个氨基酸，另一端有一个区域，该区域识别mRNA代码中三个连续核苷酸的特定运行，即“三联体”。在蛋白质合成过程中，核糖体沿着 mRNA 三联体移动，每一步都会招募具有适当识别序列的 tRNA，核糖体将 tRNA 的氨基酸转移到不断增长的氨基酸链（即多肽）上。该项目将研究蛋白质合成的三个方面。第一个是 mRNA 如何被招募到核糖体并在亚基之间输送。在哺乳动物中，mRNA 与核糖体中分离的小亚基相互作用，并与许多其他称为起始因子的蛋白质形成复合物。该起始复合物沿着 mRNA 移动，扫描序列，直到识别出标志着蛋白质合成开始的特定三联体。此时，大亚基连接并且氨基酸聚合开始。我们希望通过纯化在沿 mRNA 扫描过程中或在起始三联体处暂停的起始复合物，详细表征该扫描复合物（称为 48S 复合物）的结构，并使用显微镜对其进行研究。我们的显微镜使用电子而不是光，并且功能非常强大，因为电子的短波长可以使精细的分子细节可视化。样品也会在 -180°C 下冷冻，使其保持自然稳定的状态，因此该技术被称为“冷冻电子显微镜”(cryo-EM)。我们获得的图像将向我们展示 48S 复合物的结构，并告诉我们各种起始因子如何允许小亚基沿着 mRNA 扫描并开始蛋白质合成。第二个方面是延伸，即通过 tRNA 将氨基酸添加到不断增长的链中。核糖体内有多个 tRNA 结合位点。 tRNA 在一个位点进入，与其互补的 mRNA 三联体结合，并将其氨基酸转移到不断增长的链上，移动到相邻位点为下一个 tRNA 腾出空间，最后通过退出位点离开核糖体。 tRNA 的运动很难在分子水平上进行分析。我们发现了一种称为假结的 mRNA 信号，它会阻止核糖体沿着 mRNA 前进，并在 tRNA 在核糖体内移动时使核糖体停滞。我们将使用冷冻电镜研究停滞在假结处的核糖体结构，以阐明 tRNA 运动的分子细节。假结还可以导致核糖体改变阅读框，即从阅读一组三联体（一个框架）转变为阅读一组重叠的（另一个框架），这会导致蛋白质中出现全新的氨基酸序列。因此，对假结停滞核糖体的冷冻电镜研究可能会为核糖体如何保持正确的阅读框架提供信息。该项目的最后一个方面涉及从哺乳动物细胞制备的核糖体的结构。由于核糖体在控制蛋白质合成速率，甚至控制哪些蛋白质的生成方面发挥着重要作用，因此在不同的细胞状态下，核糖体会以多种方式进行修饰，从而影响哪些蛋白质的生成及其速度。通过确定已知条件下生长的细胞的核糖体结构，我们将在展示活细胞中核糖体机制和调节过程之间的关系方面迈出第一步。

项目成果

Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U6A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting.

U6A 七聚体上程序化的 -1 或 -2 核糖体移码的间隔长度依赖性支持信使 RNA (mRNA) 张力在移码中的作用。

• DOI: http://dx.10.1093/nar/gks629
• 发表时间: 2012
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Lin Z

Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition.

溶素的结构揭示了成孔蛋白的共同进化起源及其鞘磷脂识别模式。

• DOI: http://dx.10.1016/j.str.2012.06.011
• 发表时间: 2012
• 期刊: 1993)
• 作者: De Colibus L

Direct observation of distinct A/P hybrid-state tRNAs in translocating ribosomes.

直接观察易位核糖体中不同的 A/P 混合状态 tRNA。

• DOI: http://dx.10.1016/j.str.2009.12.007
• 发表时间: 2010
• 期刊: 1993)
• 作者: Flanagan JF

Application of In-Fusion™ cloning for the parallel construction of E. coli expression vectors.

应用 In-Fusion™ 克隆平行构建大肠杆菌表达载体。

• DOI: 10.1007/978-1-62703-764-8_15
• 发表时间: 2024-09-13
• 期刊: Methods in molecular biology
• 作者: L. Bird;Heather Rada;J. Flanagan;J. Diprose;R. Gilbert;R. Owens
• 通讯作者: R. Owens